ABSTRACT One of the most important concepts in modern genetics is the realization that over 90% of the mammalian genome is transcribed into RNA. These non-protein coding RNAs have been described as the dark matter of the genome as the function of most of these RNAs has yet to be determined. Just over 5 years ago, we stumbled onto a novel set of tRNA derived small RNAs (tsRNAs). These tRNA derived fragments are also commonly called trfRNAs, and were originally thought to represent degradation products. However, it is now clear there are hundreds or perhaps thousands of different RNA fragments generated in mammalian cells and there is increasing evidence that these are functionally important. As a group, they have been proposed to regulate genes at many different levels. We plan to study the 3?tsRNAs (derived from the 3? end of the mature tRNA) currently the least well studied of the various types of fragments, and for which no definitive function has yet to be described. We have established that one specific RNA, the 22nt CAG-Leucine 3?tsRNA when down regulated by the addition of antisense oligonucleotides in rapidly dividing cells inhibits ribosome biogenesis by first limiting the translation of at least one ribosomal protein mRNA. This results in rapid cellular apoptosis. In contrast, the addition of a 3?tsRNA mimic increases cellular proliferation and can complement the ribosome biogenesis defects in cells. These data bring to light a previously unknown post- transcriptional gene regulatory pathway. We plan to further explore the mechanism of how this specific 3?tsRNA-mediates translational gene regulation as well as use new high-throughput sequencing approaches to identify and then test other 3?tsRNAs that may participate in similar types of gene regulation. Finally, we propose to use gene therapy and oligonucleotide antisense delivery to pursue the therapeutic potential of manipulating 3?tsRNAs in treating medical conditions that affect the liver. We believe unraveling the molecular mechanism of this regulatory circuit is important both to further understand how genes are regulated and will provide novel therapeutic targets.